SIRT1调控Nrf2/HO-1信号通路对脓毒症诱导的急性肺损伤的影响

Effect of SIRT1 regulating Nrf2/HO-1 signaling pathway on sepsis-induced acute lung injury

目的:探讨沉默信息调节子1(SIRT1)能否调控核因子E2相关因子2/血红素加氧酶-1(Nrf2/HO-1)信号通路,以及在脓毒症大鼠急性肺损伤(ALI)中的作用。方法:将24只雄性SD大鼠按随机数字表法分为假手术组(Sham组)、盲肠结扎穿孔术(CLP)致脓毒症模型组(CLP组)、脓毒症+SIRT1特异性激动剂组(CLP+SRT1720组,术前2 h腹腔注射SRT1720 10 mg/kg)、脓毒症+SIRT1特异性抑制剂组(CLP+EX527组,术前2 h腹腔注射EX527 10 mg/kg),每组6只。制模后24 h处死大鼠取肺组织,并进行肺组织病理评分(Smith评分),检测肺组织超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)、丙二醛(MDA)、8-羟基脱氧鸟苷(8-OHdG)、肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6、IL-1β)以及SIRT1、Nrf2、HO-1的mRNA和蛋白表达。结果:与Sham组相比,CLP组肺泡结构受损、肺泡间隔增宽、炎症细胞浸润、肺毛细血管充血,Smith评分显著升高;肺组织炎症因子TNF-α、IL-6、IL-1β水平均明显升高,SOD活性及GSH含量降低,MDA及8-OHdG含量升高;肺组织SIRT1、Nrf2和HO-1的mRNA及蛋白表达降低。使用SIRT1特异性激动剂后,CLP+SRT1720组较CLP组肺组织损伤得到明显缓解,Smith评分及肺组织TNF-α、IL-6、IL-1β水平明显下降〔Smith评分(分):2.83±0.75比5.67±0.52,TNF-α(ng/L):36.78±5.36比66.99±5.44,IL-6(ng/L):23.97±3.76比45.70±4.16,IL-1β(ng/L):16.76±1.39比39.64±2.59,均 P<0.05〕,SOD活性及GSH含量升高〔SOD(kU/g):115.88±3.31比101.65±1.09,GSH(μmol/g):8.42±0.81比5.74±0.46,均 P<0.05〕,MDA及8-OHdG含量降低〔MDA(μmol/g):5.24±0.33比9.86±0.66,8-OHdG(ng/L):405.76±8.54比647.12±10.64,均 P<0.05〕,肺组织SIRT1、Nrf2和HO-1的mRNA及蛋白表达明显升高〔SIRT1 mRNA(2^(-ΔΔCT)):1.49±0.15比0.64±0.03,Nrf2 mRNA(2 -ΔΔCT):1.19±0.08比0.84±0.02,HO-1 mRNA(2^(-ΔΔCT)):1.80±0.41比0.64±0.11,SIRT1蛋白(SIRT1/β-actin):1.03±0.06比0.52±0.05,Nrf2蛋白(Nrf2/β-actin):1.14±0.10比0.63±0.05,HO-1蛋白(HO-1/β-actin):1.01±0.11比0.73±0.03,均 P<0.05〕。而使用SIRT1特异性抑制剂后,CLP+EX527组较CLP组肺组织损伤明显加重,Smith评分及肺组织TNF-α、IL-6、IL-1β水平明显升高〔Smith评分(分):8.00±0.89比5.67±0.52,TNF-α(ng/L):87.15±4.23比66.99±5.44,IL-6(ng/L):66.79±2.93比45.70±4.16,IL-1β(ng/L):58.99±2.12比39.64±2.59,均 P<0.05〕,SOD活性及GSH含量降低〔SOD(kU/g):72.84±3.85比101.65±1.09,GSH(μmol/g):3.30±0.67比5.74±0.46,均 P<0.05〕,MDA及8-OHdG含量升高〔MDA(μmol/g):14.14±0.70比9.86±0.66,8-OHdG(ng/L):927.66±11.47比647.12±10.64,均 P<0.05〕,肺组织SIRT1、Nrf2和HO-1的mRNA及蛋白表达明显降低〔SIRT1 mRNA(2^(-ΔΔCT)):0.40±0.07比0.64±0.03,Nrf2 mRNA(2^(-ΔΔCT)):0.48±0.07比0.84±0.02,HO-1 mRNA(2^(-ΔΔCT)):0.27±0.14比0.64±0.11,SIRT1蛋白(SIRT1/β-actin):0.20±0.05比0.52±0.05,Nrf2蛋白(Nrf2/β-actin):0.45±0.01比0.63±0.05,HO-1蛋白(HO-1/β-actin):0.36±0.08比0.73±0.03,均 P<0.05〕。 结论:在脓毒症ALI大鼠模型中,可通过激活SIRT1调控Nrf2/HO-1信号通路活化,上调下游抗氧化酶的表达,减少氧化应激损伤,进而减轻脓毒症诱导的ALI。

Objective To investigate whether silence information regulator 1(SIRT1)could regulate nuclear factor E2-related factor 2/heme oxygenase 1(Nrf2/HO-1)signaling pathway and its role in acute lung injury(ALI)in sepsis rats.Methods Twenty-four male Sprague-Dawley(SD)rats were randomly divided into sham operation group(Sham group),cecal ligation and puncture(CLP)induced sepsis group(CLP group),sepsis+SIRT1 specific agonist group(CLP+SRT1720 group,10 mg/kg SRT1720 was intraperitoneally injected 2 hours before CLP),sepsis+SIRT1 specific inhibitor group(CLP+EX527 group,10 mg/kg EX527 was intraperitoneally injected 2 hours before CLP),with 6 rats in each group.The rats were killed 24 hours after modeling and their lung tissues were taken for pathological score(Smith score),superoxide dismutase(SOD),glutathione(GSH),malondialdehyde(MDA),8-hydroxydeoxyguanosine(8-OHdG),tumor necrosis factor-α(TNF-α),interleukins(IL-6,IL-1β),and SIRT1,Nrf2 and HO-1 mRNA and protein expression were detected.Results The lung tissue of the CLP group mice was severely damaged,the alveolar interval was widened and a large number of inflammatory cells infiltrated,and there was visible pulmonary capillary hyperemia.The Smith score,the levels of TNF-α,IL-6,IL-1β,MDA and 8-OHdG were significantly increased,the levels of SOD,GSH,SIRT1,Nrf2 and HO-1 were significantly decreased in CLP group.After using SIRT1 specific agonist,the lung injury in CLP+SRT1720 group was significantly alleviated compared with that in CLP group,Smith score and lung tissue TNF-α,IL-6,and IL-1βlevels were significantly decreased[Smith score:2.83±0.75 vs.5.67±0.52,TNF-α(ng/L):36.78±5.36 vs.66.99±5.44,IL-6(ng/L):23.97±3.76 vs.45.70±4.16,IL-1β(ng/L):16.76±1.39 vs.39.64±2.59,all P<0.05],SOD activity and GSH content increased[SOD(kU/g):115.88±3.31 vs.101.65±1.09,GSH(μmol/g):8.42±0.81 vs.5.74±0.46,both P<0.05],MDA and 8-OHdG contents decreased[MDA(μmol/g):5.24±0.33 vs.9.86±0.66,8-OHdG(ng/L):405.76±8.54 vs.647.12±10.64,both P<0.05],the mRNA and protein expressions of SIRT1,Nrf2 and HO-1 were increased[SIRT1 mRNA(2-ΔΔCT):1.49±0.15 vs.0.64±0.03,Nrf2 mRNA(2^(-ΔΔCT)):1.19±0.08 vs.0.84±0.02,HO-1 mRNA(2^(-ΔΔCT)):1.80±0.41 vs.0.64±0.11,SIRT1 protein(SIRT1/β-actin):1.03±0.06 vs.0.52±0.05,Nrf2 protein(Nrf2/β-actin):1.14±0.10 vs.0.63±0.05,HO-1 protein(HO-1/β-actin):1.01±0.11 vs.0.73±0.03,all P<0.05].The lung injury in CLP+EX527 group was more severe than that in CLP group,Smith score and lung tissue TNF-α,IL-6,IL-1βlevels were significantly increased[Smith score:8.00±0.89 vs.5.67±0.52,TNF-α(ng/L):87.15±4.23 vs.66.99±5.44,IL-6(ng/L):66.79±2.93 vs.45.70±4.16,IL-1β(ng/L):58.99±2.12 vs.39.64±2.59,all P<0.05],SOD activity and GSH content decreased[SOD(kU/g):72.84±3.85 vs.101.65±1.09,GSH(μmol/g):3.30±0.67 vs.5.74±0.46,both P<0.05],the contents of MDA and 8-OHdG were increased[MDA(μmol/g):14.14±0.70 vs.9.86±0.66,8-OHdG(ng/L):927.66±11.47 vs.647.12±10.64,both P<0.05],the mRNA and protein expressions of SIRT1,Nrf2 and HO-1 were decreased[SIRT1 mRNA(2^(-ΔΔCT)):0.40±0.07 vs.0.64±0.03,Nrf2 mRNA(2^(-ΔΔCT)):0.48±0.07 vs.0.84±0.02,HO-1 mRNA(2^(-ΔΔCT)):0.27±0.14 vs.0.64±0.11,SIRT1 protein(SIRT1/β-actin):0.20±0.05 vs.0.52±0.05,Nrf2 protein(Nrf2/β-actin):0.45±0.01 vs.0.63±0.05,HO-1 protein(HO-1/β-actin):0.36±0.08 vs.0.73±0.03,all P<0.05].Conclusions In the rat model of ALI induced by sepsis,SIRT1 can regulate the activation of Nrf2/HO-1 signaling pathway,upregulate the expression of downstream antioxidant enzymes,reduce oxidative stress injury,and then alleviate the ALI induced by sepsis in rats.